amiRNA technology has many advantages over more traditional
antisense or hairpin methods of gene silencing. First, the
region used for silencing is only 21 nucleotides long, thereby increasing
specificity for the gene of interest and reducing the likelihood
of inadvertently silencing nontarget genes (e.g., those that
share stretches of sequence similarity with the target gene). Silencing
of nontarget genes can be problematic when large
stretches of sequence are used for hairpin-based silencing approaches.
Second, the cloning to make the amiRNA cassette is
done by a series of straightforward site-directed mutagenesis PCRs
on an existing template plasmid. Inserts from the resulting plasmid
can then be transferred to a binary plant transformation
vector either using Gateway technology or traditional cloning approaches.
Finally, there is an easy to use web-based tool to select
optimal 21 nucleotide regions and to design the primers to be
used for the cloning